Selenophosphate (SeP), the energy-rich Se compound required for synthesis of many selenoenzymes is formed by selenophosphate synthetase (SPS) from ATP and an inorganic form of Se. The participation of specialized selenium delivery proteins to furnish Se specifically to SPS serves to maintain intracellular levels of selenium below toxic levels. Certain classes of delivery proteins that can utilize selenocysteine as substrate, selenocysteine lyases, include three NifS related proteins from E. coli and one from Methanococcus vannielii, an anaerobic organism that is particularly rich in selenoenzymes and factors required for selenoprotein biosynthesis. A selenium-binding protein that reacts with inorganic selenium also was isolated from M. vannielii. The gene encoding this protein was isolated, cloned, and expressed in E. coli by Dr. William Self. The expressed protein, obtained in good yield and identical to native protein, binds selenite. Antibodies to this protein allow its ready isolation from M. vannielii crude extracts. To date no stabilized forms or adducts of the highly oxygen-labile selenophosphate have been found either in mammalian or bacterial systems. Either this general selenium donor compound must be synthesized in situ wherever needed or some transportable form should exist. Based on properties of a thiophosphate-menadione addition product that we prepared previously, a corresponding derivative of selenophosphate might exist. The possibility that a unique menadione-dependent paranitrophenyl phosphatase could have a role in its formation is being examined by Dr. Lara Campbell. This enzyme is relatively abundant in certain selenium-rich anaerobic bacteria and has no known biological function. As a continuation of studies on human thioredoxin reductase, Dr. Shoshana Bar-Noy tested the feasibility of producing C-terminal extensions to allow separation of the active selenocysteine-containing enzyme from prematurely terminated inactive forms. Unfortunately, Dr. Bar-Noy completed her allowed stay at NIH and left the laboratory on June 1, 2002, without finishing the project. A group of selenium-dependent molybdopterin hydroxylases present in some anaerobic bacteria contain an unidentified cofactor form of selenium. A purine hydroxylase discovered and studied by Dr. William Self is an excellent system for study of this selenium cofactor, particularly as regards its mode of synthesis and mechanism of addition to these enzymes. Model donor compounds, such as a lipoic acid-selenium adduct, are prepared by Dr. Self and tested. A collaborative research effort initiated by Dr. Gerard Lacourciere on various selenium delivery proteins that are detected as bacterial gene products by other investigators was continued by Dr. Matt Wolfe after Dr. Lacourciere left NIH on April 12, 2002. These are characterized as to ability to furnish atomic selenium for selenophosphate synthetase and types of interactions with the enzyme.